Earlier findings in experimental animals have indicated the presence of nuclear and nucleolar antigens in tumors which were not found in non-tumor tissues (R. K. Busch et al, Cancer Res. 34, 2362, 1974; Yeoman et al, Proc. Natl. Acad. Sci. US 73, 3258, 1976; Busch and Busch, Tumori 63, 347, 1977; Davis et al, Cancer Res. 38, 1906, 1978; Marashi et al, Cancer Res. 39, 59, 1979). In these early studies by the inventors, antibodies were prepared to nucleoli of rat normal and neoplastic cells by immunization of rabbits (R. K. Busch et al, supra; Busch and Busch, supra; Davis et al, supra). Bright nucleolar fluorescence was demonstrated in the acetone-fixed tumor cells by the indirect immunofluorescence method. It was also found that the immunoprecipitin bands in Ouchterlony gels formed with antisera to Novikoff hepatoma nucleolar antigens extracted from rat Novikoff hepatoma nucleoli differed from the corresponding immunoprecipitin bands produced with liver nucleolar antigens and antiliver nucleolar antisera (Busch and Busch, supra).
Further specificity was shown when antitumor nucleolar antiserum absorbed with liver nuclear extracts produced positive nucleolar fluorescence in Novikoff hepatoma ascites cells but not in liver cells. Conversely, antiliver nucleolar antiserum absorbed with tumor nucleolar extracts did not produce detectable tumor nucleolar fluorescence but did produce positive fluorescence in liver nucleoli (Davis et al, supra).
Inasmuch as immunofluorescence analysis indicated that differences were observable in acetone-fixed tumor smears and normal rat cell smears (particularly after absorption of the antisera with normal liver nuclei and nucleoli), attempts were made to utilize these antisera to rat tumor nucleolar antigens in testing corresponding tissue samples derived from human tumors. Studies with antibodies to rodent tumor nucleoli showed that positive immunofluorescence was not found in human tumor nucleoli. In view of this, the present inventors began a new series of experiments to find specific human nucleolar antigens. Positive immunofluorescence was then found in human tumor tissues with antisera and antibodies to these new human tumor nucleolar preparations. In these studies, the antibodies were absorbed with placental nuclear sonicates as well as fetal calf serum (Busch et al, 39, 3024, 1979; Davis et al, Proc. Natl. Acad. Sci. USA 76, 892, 1979; Smetana et al, Life Sci. 25, 227, 1979).
The present invention has resulted from studies designed to produce monoclonal antibodies to specific human tumor nucleolar antigens. The advantage of monoclonal antibodies over polyclonal antibodies is that monoclonal antibodies are highly specific and they can be harvested in an unlimited supply. Such monoclonal antibodies have now been developed to a human tumor nucleolar antigen (p145).
The nucleolar antigen (p145) has a molecular size of 145 kD and has been shown by immunoelectromicroscopy to be localized within fibrillar centers and granular region of the nucleous of tumor cells. Monoclonal antibodies to the nucleolar antigen p145 have been used in immunofluorescence and/or immunoperoxidose methods for determining the expression of this antigen in various pathologic and normal human tissues.
The following Table I presents a summary of the human cancers in which bright nucleolar immunofluorescence was found with monoclonal antibodies to nucleolar antigen p145. These studies support the previous studies using polyclonal antisera that human tumors contain common nucleolar antigen(s) that can be detected with specific antibodies.
In the non-tumor tissues, benign tumors, and inflammation states, negative results were generally obtained as indicated in the following Table II.
These results, originally obtained with immunofluorescence, have been verified and extended with immunoperoxidase methods.